EpiQuik™ HDAC8 Assay Kit
Contents
1. EpiQuik HDAC8 Assay Kit Base Catalog P 4007 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik HDAC8 Assay Kit is very suitable for measuring HDAC8 levels from various fresh tissues and cultured mammalian cells 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Dameron seve KIT CONTENTS Components 48 assays 96 assays P 4007 48 P 4007 96 HC1 10X Wash Buffer ll ml 22 ml HC2 HDAC Assay Buffer 1 ml 2 ml HC3 Blocking Buffer 10 ml 20 ml HC4 Capture Antibody 200 ug ml 13 ul 26 ul HC5 Detection Antibody 200 ug ml 10 ul 20 ul HC6 Developing Solution 6 ml 12 ml HC7 Stop Solution 3 ml 6 ml HDAC8 Control 100 ng ul 12 ul 24 ul 8 Well Assay Strip with Frame 6 12 User Guide For maximum recovery of the products centrifuge the original vial after thawing prior to opening the cap SHIPPING amp STORAGE The kit is shipped in two parts one part at ambient room temperature and the second part on frozen ice packs at 4 C Upon receipt 1 Store HC5 and HDAC8 Control at 20 C 2 Store HC1 HC3 HC4 HC6 and 8 Well Assay Strips at 4 C away from light 3 Store HC2 and HC7 at room temperature The kit is stable for up to 6 months from the shipment date when stored properly Note Check if wash buffer2. 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Ensure sufficient amount of positive control is added Follow the guidance in the protocol for storage of positive control Ensure extract contains sufficient amount of proteins Ensure the nuclear extracts are stored at 80 C Page 5 Printed 2014 03 03 P 4007 High Background Present for the Blank The well is not washed enough Check if wash at each step is performed according the protocol Contaminated by the positive Ensure the well is not contaminated by adding control positive control accidentally or by using positive control contaminated tips Overdevelopment Decrease development time in Step 10 RELATED PRODUCTS P 4002 EpiQuik HDAC Activity Inhibition Assay Kit Colorimetric P 4005 EpiQuik HDAC1 Assay Kit P 4006 EpiQuik HDAC2 Assay Kit 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 201109 03 P 4007
3. HC1 contains salt precipitates before using If so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED O Orbital shaker O Pipettes and pipette tips O Microplate reader O 1 5 ml microcentrifuge tubes GENERAL PRODUCT INFORMATION Quality Control Epigentek guarantees the performance of all products in the manner described in our product instructions Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 2 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 03 03 Epigentek Group Inc All rights reserved Products are for research use only P 4007 Usage Limitation The EpiQuik HDAC8 Assay Kits are for research use only and are not intended for diagnostic or therapeutic application Intellectual Property EpiQuik is a trademark of Epigentek Group Inc A BRIEF OVERVIEW Histone deacetylases HDACs play a critical role in transcriptional repression of the gene expression in eukaryotic cells through catalyzing the hydrolytic removal of acetyl groups from histone lysine residues HDACs are tightly involved in cell cycle regulation cell proliferation and in development of human cancer HDAC inhibition displays signiticant effects on apoptosis cell cycle arrest and differentiati
4. rights reserved Products are for research use only P 4007 HDAC8 level OD ml sample OD blank OD x sample dilution For accurate calculation plot OD value versus amount of HDAC8 control and determine the slope as delta OD ng Calculate the amount of HDAC8 using the following formula Amount ng mg protein Slope X Protein amount ug Nuclear extract added into sample wells at Step 3 TROUBLESHOOTING OD sample blank x 1000 No Signal for Both the Positive Control and the Samples Reagents are added incorrectly The well is not completely dried The well is incorrectly washed before protein coating Incubation time and temperature are incorrect Check if reagents are added in order and if some steps of the procedure are omitted by mistake Ensure the well is incubated with no humidity and dry before adding block buffer Ensure the well is not washed before adding positive control or protein extracts Ensure the incubation time and temperature described in the protocol are correctly followed No Signal or Very Weak Signal for Only the Positive Control The positive control is insufficiently added to the well The positive control degraded due to incorrect storage No Signal for Only the Sample The protein amount is added into well insufficiently Nuclear extracts are incorrectly stored 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax
5. an assay with this kit the nuclear proteins containing HDAC8 are stably coated on the strip wells The HDAC8 is recognized with high affinity specific antibody The amount of HDAC8 can be quantitied through HRP conjugated secondary antibody color development system and is proportional to the intensity of color development Page 3 Printed 2014 03 03 P 4007 ij Nuclear extract Incubate with substrate and assay buffer for 60 min Add capture antibody Add detection antibody Add developing solution for developing color and measure absorbance fe FFA Ff Schematic Procedure for Using the EpiQuik HDAC8 Assay Kit PROTOCOL l Prepare nuclear extracts by using you own successful method For your convenience and best results Epigentek offers a nuclear extract kit Cat OP 0002 1 optimized for use in EpiQuik series Nuclear extracts can be used immediately or stored at 80 C for future use 2 Determine number of the strip wells required the strip wells can be broken off Leave these strip wells in the plate frame remaining unused strips can be put back in the bag Seal the bag tightly and store at 4 C Dilute 10 X HC1 with distilled water pH 7 2 7 5 to the 1 X HC1 3 Adjust protein concentration to 0 4 1 ug ul with HC2 and add 5 ul 2 5 ug of the protein solution into central area of each well Spread out the solution over the bottom of the strip well by pipetting the solution up and down several times Incubate the
6. on in cancer cells HDAC inhibitors are currently being developed as potential anticancer agents Three distinct families of HDACs have been described comprising a group of at least 20 proteins in humans HDAC8 is a class histone deacetylase containing 377 amino acid residues HDAC8 has been shown to interact directly with transcription factors and has been shown to deacetylate histone proteins H3 and H4 The major assay for measuring the expression or amount of HDAC8 protein currently is the Western blot This method requires electrophoresis and transfer process which makes the assay inconvenient time consuming and has low throughput The EpiQuik HDAC8 Assay Kit addresses these problems by using a unique procedure to measure amount of HDAC8 The kit has following features e The fastest procedure which can be finished within 3 hours e Innovative colorimetric assay to semi quantitatively measure HDAC8 amount with no need of electrophoresis e Strip microplate format makes the assay flexible manual or high throughput analysis e Simple reliable and consistent assay conditions PRINCIPLE amp PROCEDURE 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only The EpiQuik HDAC8 Assay Kit is designed for measuring total HDAC8 amount from tissues or cells In
7. strip wells at 37 C with no humidity for 60 90 min to evaporate the solution and completely dry the wells For the blank add 5 ul of HC2 to the wells For the positive control dilute HDAC8 control to 2 30 ng ul with HC2 and add 5 ul 10 150 ng of diluted HDAC8 control solution to the wells 4 Add 150 ul of HC3 to the dried wells and incubate at 37 C for 30 45 min 5 Aspirate and wash each well with 150 ul of 1 X HC1 three times 6 Dilute the HC4 at the 1 200 ratios to 1 ug ml with 1 X HC1 Add 50 ul of diluted HC4 to each assay strip well and 2 control strip wells Incubate the samples at room temperature for 60 min on a orbital shaker 50 100 rpm 7 Aspirate and wash each well with 150 ul of 1 X HC1 four times 8 Dilute the HC5 at the 1 1000 ratios to 0 2 ug ml with 1 X HC1 Add 50 ul of diluted HC5 to each strip well and incubate at room temperature for 30 min 9 Aspirate and wash each well with 150 ul of 1 X HC1 four times Allow 3 min for last wash 10 Add 100 ul of HC6 to each well and incubate at room temperature for 2 10 min away from light Monitor color development in the sample and standard well blue 11 Add 50 ul of HC7 to each well and read absorbance on microplate reader at 450 nm 12 Calculate HDAC8 level 110 Bi County Blvd Ste 122 Farmingdale NY 11735 i i Page 4 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 03 03 Epigentek Group Inc All
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